Acute kidney injury (AKI) due to renal ischemia and reperfusion (IR) is a major clinical problem without effective therapy. It is estimated that ~$10 billion per year is spent treating AKI in the United States. Exciting and novel preliminary data generated for this proposal suggest that A1AR activation stimulates sphingosine kinase-1 (SK1) enzyme activity in renal proximal tubule and endothelial cells to increase sphingosine 1- phosphate (S1P) generation. Additional preliminary data suggest a crucial role of SK1 and S1P1 receptors in mediating the renal protective effects following A1AR activation. These findings mechanistically link together for the first time two cytoprotective signaling pathways (A1AR and SK1) leading us to hypothesize that A1ARs protect against renal IR via phosphorylation as well as upregulation of SK-1 in renal proximal tubule and endothelial cells. We hypothesize that enhanced S1P synthesis subsequently activates S1P1 receptors in renal proximal tubules, endothelial cells and T-lymphocytes infiltrating the kidney to attenuate renal IR injury. To address this hypothesis, we have formulated the following 3 aims: Aim #1: To determine that SK1 and S1P1 receptor signaling is necessary for A1AR-mediated renal protection. Aim #2: To define signaling pathways mediating A1AR-stimulation of SK1 and S1P synthesis in renal proximal tubule and endothelial cells. Aim #3: To design A1AR and SK1-S1P-based renal protective strategies against IR injury without systemic side effects of A1AR agonist treatment. To test these aims, we will utilize both in vivo (murine renal IR) and in vitro (primary cultures and immortalized proximal tubule and endothelial cell lines) models of IR injury to elucidate the mechanisms of A1AR-mediated SK1 modulation and enhanced S1P synthesis. Cre-lox mouse technology will allow us to selectively delete key signaling intermediates in specific cell types (proximal tubule, endothelial or leukocytes) to further define the role of SK1 and S1P1 receptors in A1AR-mediated renal protection. Our proposed novel research integrates whole animal, molecular, histological as well as biochemical techniques, enabling a new understanding of the mechanisms of A1AR-mediated reduction in renal inflammation, necrosis and apoptosis. This, in turn, will contribute to improved therapeutic regimens for the protection of renal function in patients. As SK1 signaling controls numerous physiological effects including tissue injury, inflammation and immune response in many organs, our findings of A1AR-mediated S1P modulation represent a significant paradigm shift with significant implications for organ protection beyond renal IR injury. PUBLIC HEALTH RELEVANCE: The central hypothesis of this proposal is that A1 adenosine receptor activation reduces all 3 components of renal injury (apoptosis, necrosis and inflammation) via stimulation of sphingosine kinase-1 and activation of sphingosine 1-phosphate 1 receptors. Specifically, we will 1) conclusively define that A1AR-mediated renal protection is dependent on SK1 and S1P signaling, 2) determine the mechanisms of A1AR-mediated SK1 activation and enhanced S1P synthesis and 3) design A1AR-based renal protective strategies against IR injury. Our studies test major novel concepts and provide potential paradigm shifts with immediate clinical translatability. Success of our aims offers a new therapeutic approach to reduce the clinical perils from AKI and have implications in organ protection strategies beyond the kidney.